In the eukaryotic nucleus, an energy source is required for remodeling chromatin at S phase and at M phase, and during the synthesis and export of RNA. Although nucleoside triphosphates fuel most processes in the cell, other high energy intermediates are involved. Prothymosin alpha, an abundant, acidic, nuclear protein, has been identified by our laboratory as energy-rich. Three lines of evidence indicate that this small, presumably unfolded mammalian protein contains unusual phosphorylated residues: 1) Prothymosin alpha continues to be metabolically labeled with [32P]orthophosphate despite mutations at the Ser and Thr residues previously thought to be the sites of substoichiometric phosphorylation. 2) Immediately upon cell lysis, the pH stability curve of metabolically-labeled native [32P]prothymosin alpha resembles that of acetyl phosphate. 3) After a brief incubation at pH 7, the stability pattern changes to one characteristic of phosphoesters, an observation consistent with transfer of phosphate to stable positions in vitro. Further analysis using COS cells lysed in the presence of sodium borohydride revealed the following: phosphate decreases 8-fold in the presence of the reducing agent; 4-8 Glu residues per molecule vanish; tritium from [3H]NaBH4 is introduced into proline, a product derived from the reductive cleavage of phosphoglutamate; and a peptide with homology to the histone binding site of nucleoplasmin, a chromatin remodeling protein found in Xenopus laevis, is the sole repository of the label. The conclusion was drawn that phosphorylation of prothymosin alpha occurs solely on glutamate residues resulting in stoichiometric levels of high energy phosphoanhydride bonds. This is the first report of protein-bound glutamyl phosphates in mammals. Several methods for the determination of the half-life of prothymosin alpha's glutamyl phosphates in vivo have been developed. With these techniques, we showed that glutamyl phosphate turnover is metabolically regulated and that phosphates on under-utilized prothymosin alpha molecules have enhanced stability. Upon the cessation of transcription, but not during S phase, prothymosin alpha phosphate survival increases to values found in much longer lived, small, acyl phosphates in aqueous solution. Our data are consistent with a role for utilization of prothymosin alpha's phosphates in the remodeling of transcriptionally active chromatin.